Probing concentration-dependent behavior of DNA-binding proteins on a single-molecule level illustrated by Rad51
Journal article, 2013

Low throughput is an inherent problem associated with most single-molecule biophysical techniques. We have developed a versatile tool for high-throughput analysis of DNA and DNA-binding molecules by combining microfluidic and dense DNA arrays. We use an easy-to-process microfluidic flow channel system in which dense DNA arrays are prepared for simultaneous imaging of large amounts of DNA molecules with single-molecule resolution. The Y-shaped microfluidic design, where the two inlet channels can be controlled separately and precisely, enables the creation of a concentration gradient across the microfluidic channel as well as rapid and repeated addition and removal of substances from the measurement region. A DNA array stained with the fluorescent DNA-binding dye YOYO-1 in a gradient manner illustrates the method and serves as a proof of concept. We have applied the method to studies of the repair protein Rad51 and could directly probe the concentration-dependent DNA-binding behavior of human Rad51 (HsRad51). In the low-concentration regime used (100 nM HsRad51 and below), we detected binding to double-stranded DNA (dsDNA) without positive cooperativity. (C) 2013 Elsevier Inc. All rights reserved.

GENERATION

MECHANISMS

DNA

SUPPORTED LIPID-BILAYERS

DIFFUSION

Supported lipid bilayer

Microfluidics

GRADIENTS

HOMOLOGOUS RECOMBINATION

Single molecule

Fluorescence microscopy

Rad51

STRANDED-DNA

FILAMENTS

Author

Karolin Frykholm

University of Gothenburg

Camilla Freitag

University of Gothenburg

Fredrik Persson

University of Gothenburg

J. O. Tegenfeldt

University of Gothenburg

Annette Graneli

University of Gothenburg

Analytical Biochemistry

0003-2697 (ISSN) 1096-0309 (eISSN)

Vol. 443 2 261-268

Subject Categories

Biochemistry and Molecular Biology

DOI

10.1016/j.ab.2013.08.023

More information

Created

10/10/2017